SPECIFICATION OF TISSUE-RESIDENT MACROPHAGES DURING ORGANOGENESIS

ELVIRA MASS, IVAN BALLESTEROS, MATTHIAS FARLIK, FLORIAN HALBRITTER, PATRICK GÜNTHER, LUCILE CROZET, CHRISTIAN E. JACOME-GALARZA, KRISTIAN HÄNDLER, JOHANNA KLUGHAMMER, YASUHIRO KOBAYASHI, ELISA GOMEZ-PERDIGUERO, JOACHIM L. SCHULTZE, MARC BEYER, CHRISTOPH BOCK, FREDERIC GEISSMANN

Journal: ScienceIF: 34.661Published on: 4th August 2016Citation: E. Mass et al., Science 10.1126/science.aaf4238 (2016)

Akansha GangulyMB-0415MBC 404

16th Dec 2016Department of Biotechnology

CONTENTS

INTRODUCTION MATERIALS AND METHODS RESULTS AND DISCUSSION CONCLUSIONS REFERENCES

INTRODUCTION

Embryonic development and tissue homeostasis = cooperation between specialized cell types.

Resident macrophages – professional phagocytes that survey surroundings, eliminate unfit cells, microorganisms and metabolic waste, produce a large range of bioactive molecules and growth factors.

Tissue-specific purposes: (examples)Microglia in central nervous system (neuronal circuit development), Kupffer cells (scavenge blood particles and dying red blood cells in the liver), Alveolar macrophages (uptake surfactant, remove airborne pollutants from the airways). Resident macrophage diversity based on tissue-specific gene expression profiles

(responses to specific cues from their microenvironment, different developmental processes, and the contribution of distinct progenitors cell types)1-3.

Preferential expression of transcription factors in macrophage subsets : Gata6 (large peritoneal macrophages)4, Runx3 (Langerhans cells)5, Nr1h3 (splenic marginal zone macrophages)6, SpiC (splenic red pulp macrophages)7, Pparγ (alveolar macrophages)8.

Rationale:1. Tissue-resident macrophages originate partly from mesodermal Erythro-Myeloid

Progenitors (EMP) yolk sac: invade the embryo proper at the onset of organogenesis, forming progenitor macrophages (pMacs).

2. Tissue-resident macrophages self-maintained in postnatal tissues, independently of definitive hematopoietic stem cells (HSCs) in a steady state. We therefore

3. Hypothesis : resident macrophages represent a ‘founding’ cell type within most organ anlagen. Generation of macrophage diversity may be integral to organogenesis.

MATERIALS AND METHODS

Genotyping of conditional knockout mice for Csf1riCre, Csf1rMeriCreMer, Id3−/−; Id1fl/fl , Tnfrsf11aCre

Preparation of cell suspensions and cell sorting (dispase degradation and immunostaining) Immunofluorescence, microscope imaging, analysis of cytokine (CD45) rich areas of

sections Generation and analysis of Kupffer cells in Id3−/− mice and gene sequencing (qRT-PCR for

ld3, Nr1h3, GAPDH, gene sequencing and mapping on mouse genome, expression analysis) Generation and analysis of ‘bulk’ transcriptomes from candidate EMP, pMac, and

macrophage populations from E9 to P21 (RNA isolation, library construction, sequence analysis, evaluation from RNA-seq transcriptome data)

Generation and analysis of single cell transcriptomes (MARS-Seq, cDNA reverse transcription, library sequencing, generation and enrichment of tissue signatures)

RESULTS AND DISCUSSIONFig. 1 A core macrophage program is initiated simultaneously in pMacs in all tissues.(A) Summary of surface phenotype used

for EMPs, pMacs, and macrophages. (B) Scorecard visualization of

differentially upregulated genes in pMacs (E9.5 and E10.25) in comparison to EMPs, based on whole transcriptome sequencing of indicated populations. Relative enrichment of differentially upregulated genes in pMacs across cell types and tissues (y-axis) and developmental time points (x-axis, from E9 to P21).

(C) Stained preparations of sorted EMPs, pMacs and early macrophages from yolk sac (YS), head, limbs and fetal liver (FL) at E10.25 and E12.5.

(D) Plot of single cell RNA-seq data showing distribution of CD45low/+ cells from E10.25 embryos into three clusters

(E) Superimposition of EMP-, pMac-, or macrophage-specific signatures defined by the bulk RNA sequencing on (D)

(F) (D) overlaid with the relative expression values for Kit and Maf.

Fig. 2 Tissue colonization by pMacs is Cx3cr1-dependent. (A) Expression of GFP and

Dectin-1 in Cx3cr1gfp/+ mice during development (E8.5-10.5) in Kit+ cells (CD45low, Kit+), pMacs and macrophages. sp: somite pairs.

(B) Flow cytometry analysis in Cx3cr1+/− and Cx3cr1−/− of pMacs and macrophages from yolk sac (YS), head, and caudal at E9.5 (upper panel) and liver, YS, head, and limbs at E10.5 (lower panel). Circles represent individual mice. sp: somite pairs.

Fig. 3 Early specification of tissue-resident macrophages. (A)Flow cytometry analysis of liver, brain, lung and skin F4/80+ cells from postnatal mice (4 weeks old) showing expression of TFs and cytokines. Black dotted line depicts expression by all macrophages (CD45+ CD11blowF4/80high) and green tinted line shows expression by the subsets of YFP+ macrophages. Gray histograms show the fluorescence intensity of the FMO controls for all macrophages and YFP+ macrophages.(B and C) Scorecard analysis of all differentially upregulated genes in postnatal macrophages from bulk RNA-seq. The scorecards show the relative enrichment of each set of upregulated genes across each cell type (y-axis) and developmental time point (x-axis). (D) Heatmap representing all differentially upregulated transcriptional regulators from bulk RNA-seq.

Transcription factor ld3 important for Kupffer cell development-1. E10.25 Id3-deficient embryos had normal or increased numbers of pMacs/early macrophages in the YS, but macrophages were reduced in numbers in the embryo proper (liver and head) in comparison with littermate controls. Development of microglia and kidney macrophages appeared normal.2. RNA-seq analysis of Id3−/− adult Kupffer cells indicated the upregulation (>3-fold) of Id1 expression, and expression analysis evidenced that Id3−/− cells overexpressed genes involved in the control of cell death and cytokine responses and down-regulated genes involved in metabolic processes.

CONCLUSIONS

EMPs rapidly differentiate into pMacs, simultaneously colonize the whole embryo from E9.5 in a Cx3cr1-dependent manner while differentiating into macrophages.

pMacs do not yet have macrophage morphology but are in the process of establishing a full core macrophage differentiation program that includes cytokine receptors, phagocytic and pattern recognition receptors, and complement.

Establishment of a core macrophage differentiation program in EMP-derived pMacs, is followed by the initiation of macrophage specification via the acquisition of tissue-specific transcriptional regulators, such as Id3 in Kupffer cells (E8.5 to E10.5).

Differentiation of resident macrophages is a developmental process and an integral part of organogenesis, independent of postnatal changes in the environment in kidney, liver, and brain, but not in skin and lung.

Framework to analyze and understand the consequence(s) of genetic variation for macrophage contribution to disease pathogenesis in different tissues.

Fig. 4 Graphic summary of the establishment of the core macrophage program and subsequent specification. Cx3cr1 is expressed in pMacs and is important for colonization of the embryo. Id3 is a liver macrophage-specific gene, which is essential for Kupffer cell development.

REFERENCES

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2. Gosselin D, et al. Environment drives selection and function of enhancers controlling tissue-specific macrophage identities. Cell. 2014;159:1327–1340

3. Okabe Y, Medzhitov R. Tissue-specific signals control reversible program of localization and functional polarization of macrophages. Cell. 2014;157:832–844

4. Rosas M, et al. The transcription factor Gata6 links tissue macrophage phenotype and proliferative renewal. Science. 2014;344:645–648

5. Fainaru O, et al. Runx3 regulates mouse TGF-β-mediated dendritic cell function and its absence results in airway inflammation. EMBO J. 2004;23:969–979

6. N A-G, et al. The nuclear receptor LXRα controls the functional specialization of splenic macrophages. Nature Immunology. 2013;14:831–839

7. Kohyama M, et al. Role for Spi-C in the development of red pulp macrophages and splenic iron homeostasis. Nature. 2009;457:318–321

8. Gautier EL, et al. Systemic analysis of PPARγ in mouse macrophage populations reveals marked diversity in expression with critical roles in resolution of inflammation and airway immunity. J Immunol. 2012;189:2614–2624

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